Extended shelf life and bulk transport of perishable organic liquids with low pressure carbon dioxide

ABSTRACT

Carbon dioxide is dissolved in perishable liquids loaded into pressure vessels that are provided with low carbon dioxide head pressure so as to improve product shelf life, thereby providing options for more economical shipment, as by rail and ocean vessels and for extended transport by truck and to facilitate extended storage of perishable products and to avoid the necessity of multiple treatments for pathogen reduction.

The present application claims priority to the May 21, 2004 filing dateof U.S. provisional patent application, Ser. No. 60/573,072.

FIELD OF THE INVENTION

The present invention relates to techniques to extend the shelf life andfacilitate the bulk transport of perishable organic liquids whereby theliquids are mixed with carbon dioxide gas and held under carbon dioxidepressure, in order to extend the time before the liquid spoils orsustains material undesirable biological changes.

BACKGROUND OF THE INVENTION

Assuring the safety of fluid milk, related dairy products, and juiceswhile maintaining quality and increasing the shelf life of products is asignificant challenge for the food industry. Many perishable organicliquids, including juices, but especially raw milk, serve as suitablegrowth mediums for microorganisms. Benefits in distribution and organicliquid quality could be derived from reducing microbial growth.

The bulk transport of perishable organic liquids generally requires atleast one of the following: pasteurization or similar treatments toreduce, eliminate or control pathogens; rapid shipment; and in somecases, refrigeration. Each of these options imposes additional cost andor limitations. For instance, shipment by truck may be the quickesttransport time but still may not be sufficiently rapid to reach allmarkets. Shipment by rail or ocean cargo vessel is slower but moreeconomical. Refrigerated shipping costs are substantially higher thanthe cost for shipments not requiring refrigeration. Furthermore,refrigeration is not effective to adequately restrain the growth ofpsychrotrophic microorganisms capable of activity at temperatures below7° C. over sustained intervals of time. Each process of pasteurizationor similar pathogen reduction treatment imposes not only expense, butmay also negatively impact the flavor quality, nutritional content, andother sensory characteristics, such as color, of the treated organicliquid with a resulting negative market impact. Additionally,thermoduric microorganisms that are potential pathogens or causespoilage may survive the pasteurization process.

As a result of these concerns, today when arranging for the shipment offresh milk from the continental United States to Hawaii or a Caribbeanisland without significant dairy herds, there are two principal options,namely:

-   -   Milk is pasteurized before bulk shipment and is re-pasteurized        prior to local packaging for retail sale. The result is a flavor        not as fresh as with single pasteurization and a higher cost due        to multiple handling.    -   Milk is pasteurized and packaged for retail sale at or near the        origin and then shipped in refrigerated containers to the        destination. The result is higher cost and a loss of shelf life        at retail due to the transit period.

The repeated pasteurization of the first option is also particularlyundesirable because while most milk borne microorganisms are neutralizedby pasteurization, their lipolytic and proteolytic enzymes can surviveand result in undesirable lipolysis and proteolysis.

The major strategy to extend shelf life of unpasteurized perishableorganic liquids has been to provide rapid refrigeration. For instance,decreasing the storage temperature from 6° C. to 2° C. increases thetime for the psychrotrophic count to reach 10⁶ cfu (colony formingunits)/ml from 2.9 to 5 days (Griffith, 1987).

Several authors have reported on the use of unpressurized carbon dioxideas an anti-microbial agent in foods including dairy products. Theconcept of using CO₂ to inhibit the growth of unwanted microorganisms indairy products stems from the technology of modified atmospherepackaging. This method of shelf life extension has been adapted to fluiddairy products by directly injecting the inert gas (CO₂) therebyenhancing its inhibitory effect. The direct post-pasteurization additionof carbon dioxide (DAC) to neutral and acidic pH products can be used tocontrol contaminating organisms. DAC is widely used by cottage cheeseprocessors in North America. Carbon dioxide has also been shown toextend the shelf life of yogurt, to improve the keeping quality of rawmilk, and to extend the yields of cheese subsequently prepared from suchmilk. However, under specific combinations of pressure and temperature,CO₂ effectively precipitates the proteins from milk. For example, at 38°C. and pressures above 5514 kilopascals (kPa), or about 800 psi,complete precipitation of the casein proteins that give milk itsdistinctive white color results. CO₂ pressure treatments applied at apressure of only 294 kPa (about 43 psi) at 20° C. may result in caseinaggregation. Accordingly, pressurization has been avoided due topotential deleterious effects upon the treated liquids. In addition, andnot unrelatedly, there is an absence of suitable pressure vessels forpressurized bulk storage and transport of organic liquids. The studiesutilizing CO₂ pressure treatments have been principally directed topathogen reduction treatments with high CO₂ pressures as an alternativeto thermal pasteurization. Lower CO₂ pressures have not been previouslyutilized as conditions of storage and transportation to reduce microbialgrowth.

SUMMARY OF THE INVENTION

The present invention provides a method that extends the stability ofraw milk and other perishable organic liquids sufficiently to permittheir transport by rail or cargo ship, or by truck for greater distancesthan is ordinarily accomplished today; or shipment by any mode thatwould benefit the end user by being more cost effective or offering moretime to handle or package the product for human consumption or for otherfood, feed grade, pharmaceutical, or industrial use or extended storage.In furtherance of the invention, raw milk or other perishable organicliquids are preferably cooled to the greatest extent practical andinjected with carbon dioxide as they are loaded in food grade storageand transport containers that are pressure vessels according toapplicable pressure vessel codes, so that the filled containers can bepressurized with about 20 to 50 psi (138 to 345 kPa), and morepreferably about 30 to 50 psi (207 to 345 kPa), of carbon dioxide. Suchcarbon dioxide head pressure does not result in protein precipitationand maintains concentrations of sufficient parts per million in the rawmilk or other organic liquids so as to suppress pathogen growth, atleast of the most common obligate aerobic varieties, by lowering the phand by initiating deleterious intercellular activity and by surroundingthe pathogens in what is equivalent to their own output. In such astate, the pathogens' animation and reproduction ceases or is suitablyreduced so as to inhibit their growth and multiplication, which wouldotherwise result in spoiled product.

The pressurized carbon dioxide rich raw milk and other perishableorganic liquids may then be shipped or stored in the container withoutspoilage for periods of time greater than untreated and un-pressurizedproducts, provided the storage tanks are sufficiently insulated orrefrigerated to prevent excessive heating of the contents. Upondelivery, or when the milk or other organic liquid is needed forproduction, the container is unloaded and the carbon dioxide is releasedfrom the liquid by some form of agitation, stirring or mixingindependent of or in conjunction with negative (vacuum) pressure and theliquid is then processed in its usual fashion.

The present process can effectively extend the life of a wide variety ofperishable organic liquids including dairy products, vegetable juices,fruit juices, plant extracts, fungal extracts, flavoring agents, andcombinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of milk collection from dairy farmsto processing facilities as commonly practiced in the United States,showing the integration of a new carbon dioxide injection systemaccording to the present invention;

FIG. 2 a is an alternative of FIG. 1, showing the prior art Hawaii Modelfor shipping milk for extended times and distances;

FIG. 2 b is a schematic illustration showing a Hawaii Model for shippingmilk for extended times and distances;

FIG. 3 a is a top plan view of a tank container suitable for use inpracticing the invention;

FIG. 3 b is a side plan view of the tank container of FIG. 3 a.

FIG. 3 c is an end plan view of the tank container of FIG. 3 a.

FIG. 3 d is a detail drawing of the pressure relief valve of the tankcontainer of FIG. 3 a;

FIG. 3 e is a detail drawing of the air inlet used to pressurize thetank container of FIG. 3 a;

FIG. 3 f is a detail drawing of the loading/discharge valve of the tankcontainer of FIG. 3 a;

FIG. 4 is a schematic illustration of the carbon dioxide injectionsystem suitable for use when filling the transport containers withorganic liquids;

FIG. 5 depicts the loading and unloading connection with a tankcontainer suitable for use in the present invention;

FIG. 6 illustrates a food grade pump for loading or unloading transporttanks;

FIG. 7 illustrates carbon dioxide or air being used to pressurize thetransport tank;

FIG. 8 is a representative three stage filter to clean the air prior topressurization.

FIG. 9 is a schematic illustration of the carbon dioxide batchpressurization system utilized in connection with Example 1.

FIGS. 10 (a-e) are bar charts illustrating the changes in gram-negativelactose, Lactobacillus spp. and Standard Plate Count in raw milk treatedat (a) 68 kPa, (b) 172 kPa, (c) 344 kPa, (d) 516 kPa, and (e) 689 kPa ofCO₂ pressure at 6.1° C. for four days as described in Example 1.

FIG. 11 is a bar chart of total counts, thermoduric bacteria, totalcoliforms and E. coli counts in raw milk treated at 4° C. and 689 kPa ofCO₂ pressure after 4, 6, 8 and 9 days as described in Example 1.

FIG. 12 a is a chart plotting bacterial growth in raw whole milk treatedwith 2000 ppm CO₂ against an untreated but refrigerated control over 13days as described in Example 2.

FIG. 12 b is a chart plotting temperatures of the milk of Example 2 andthe daily local temperatures.

DETAILED DESCRIPTION OF THE INVENTION

As is described in this patent, milk injected with carbon dioxide andthe application of pressurized carbon dioxide in a transport vesselretards the growth of pathogens in the raw milk. Raw milk has been usedas an example in this text not to limit the usefulness of the patent butrather to explain the invention with reference to one of the morecommonly transported liquid products that is also one of the moreperishable products. The same process may be utilized to retard thegrowth of similar pathogens in other perishable organic liquids.Accordingly, the invention may also be practiced in connection withliquids such as fruit juices, wine, malt beverages, beveragepreparations, liquid eggs, feed grade bulk liquids as well aspharmaceutical or industrial grade liquids and other similar materialssusceptible to detrimental microbiological activity.

For example, the present invention is applicable to any liquid dairyproduct, including, but not limited to, cream, light cream, lightwhipping cream, heavy cream, heavy whipping cream, whipped cream,whipped light cream, sour cream, acidified sour cream, cultured sourcream, half-and-half, sour half-and-half, acidified sour half-and-half,cultured sour half-and-half, reconstituted or recombined milk and milkproducts, concentrated milk, concentrated milk products, reverse osmosis(RO) milk, ultra filtered (UF) milk, fractionated milk, whole milk,reduced fat or low fat content milk (e.g., 1% fat milk, 2% fat milk,etc.), nonfat (skim) milk, evaporated and condensed forms of whole milk,eggnog, buttermilk, cultured milk, cultured reduced fat or lowfat milk,cultured nonfat (skim) milk, yogurt, lowfat yogurt, nonfat yogurt,acidified milk,. acidified reduced fat or lowfat milk, acidified nonfat(skim) milk, low-sodium milk, low-sodium reduced fat or lowfat milk,low-sodium nonfat (skim) milk, lactose-reduced milk, lactose-reducedreduced fat or lowfat milk, lactose-reduced nonfat (skim) milk, reducedfat or lowfat milk or nonfat (skim) milk with added safe and suitablemicrobial organisms and any other milk product made by the addition orsubtraction of milkfat or addition of safe and suitable optionalingredients for protein, vitamin, or mineral fortification of milkproducts defined by governmental regulation.

The present invention is also applicable to other products derived fromdairy ingredients, including whey and whey products, caseinates,lactalbumin, cottage cheese, ice cream mix, ice milk mix, yogurt mix,shake mixes, batter mixes, and other dairy mixes, probiotic dairyproducts, including milk treated with Lactobacillus cultures orAcidophilus cultures, flavored milks, spreads, dips, sauces, eggnogs,flavored creamers, boiled custards, puddings, cheesecakes, milkshakes,smoothies, dairy shakes, and other shakes, as well as other productscontaining milk or other ingredients derived from dairy products.

The present invention is applicable to milk and milk-like productsderived from crop plants or grains, including but not limited to soy,rice, wheat, corn, and oats.

The present invention is applicable to avian eggs, including bothin-shell and liquid preparations. The present invention is alsoapplicable to products containing added nutritional components, e.g.,protein, minerals, vitamins, fat, fiber, sugars, salts, starches, aminoacids, and alcohols.

The present invention is further applicable to milk and products derivedfrom the milk of bovine species, goats and sheep.

The present invention is also applicable to water, carbonated water, andproducts containing water, as well as a variety of beverages and drinks.The present invention is also applicable to fermented foods, foodproducts, and beverages.

The present invention is also applicable to juices, extracts, liquidsupplements, and liquid pharmaceuticals derived from fresh, dried,frozen or canned plants, vegetables, fruits, grasses, yeasts, fungi, andcombinations thereof, including but not limited to juices or extractsderived from apples, apricots, pineapples, peaches, bananas, oranges,lemons, limes, grapefruit, plums, cherries, grapes, raisins, prunes,nectarines, kiwi, star fruit, papayas, mangos, blueberries, raspberries,strawberries, choke cherries, boysenberries, cranberries, lingenberries,pomegranates, melons, tomatoes, carrots, onions, garlic, celery,lettuce, cucumbers, radishes, broccoli, potatoes, sweet potatoes, yams,cauliflower, brussel sprouts, cabbage, rutabaga, corn, peas, greenbeans, yeast, including brewer's yeast, and mushrooms. The presentinvention also is applicable to blended, liquefied whole plants, fruits,vegetables, grasses, yeasts, fungi, and combinations thereof, includingbut not limited to the whole plants, fruits, vegetables, grasses,yeasts, fungi disclosed hereinabove.

The present invention may be applicable to a mixture of a liquid dairyproduct, e.g., skim milk, and one or more juices, extracts, liquidsupplements, and liquid pharmaceuticals.

The amount of vegetable, fruit, yeast, or fungal juice, or combinationthereof, in the product can be between 0.05% to 100%, preferably between0.1% to 75%.

The present invention may further be applicable to products containingany added flavoring agent, including any of the usual flavors, such as afruit flavor (natural or artificial, or both), vegetable flavor,chocolate flavor, vanilla flavor, and any of the usual soft drinkflavors, such as the cola flavor, ginger ale flavor, etc., or atraditional malt flavor.

The term “shelf life” is defined as the amount of time a product remainsacceptable for organoleptic, nutritional, and/or safety purposes, forthe consumer or the retailer.

The term “undesirable biological changes” includes changes in the liquidor product such that the liquid or product is unacceptable fororganoleptic, nutritional, and/or safety purposes, for the consumer.These changes may include, but are not limited to, changes in the color(brown color), decreases in the flavor quality (cooked flavor), anddecreases in the nutritional content (i.e., vitamin loss or proteinprecipitation).

The term “liquid” is defined as being a fluid or semi-fluid, e.g., apourable or flowable substance intended for human or animal consumption.

The terms “pathogens” and “food pathogens” are defined to includemicroorganisms, bacteria, viruses, and fungi, including but not limitedto psychrotrophic bacteria; lipolytic psychrotrophic bacteria;proteolytic psychrotrophic bacteria; mesophylic bacteria; Bacillusspecies, including B. cereus; Clostridium species, including C.perfringens and C. botulinum; Cryptosporidium species; Campylobacteriaspecies, including C. jejuni; Listeria species, including L.monocytogenes; Escherichia species, including E. coli and pathogenic E.coli strains; Mycobacterium species, including M. paratuberculosis;Pseudomonas species, including P. fluorescens; Helicobacteria species;Yersinia species, including Y. entercolitica; Arcobacter species;Aeromonas species; Toxoplasma species, including T. gondii;Streptococcus species; Staphylococcus species, including S. aureus;Shigella species; Salmonella species, including S. enteritidis, S.Montevideo, S. typhimurium; Cyelospora species, including C.cayetanensis; Cignatera species; Vibrio species; Plesiomonas species;Entamoeba species, including E. histolytica; Hepatitis viruses;Astroviruses; Calciviruses; enteric Adenoviruses; Parvoviruses; andRotaviruses.

CO₂ is a ubiquitous environmental bacterial stress. In accord with thepresent invention, purified CO₂ may be safely and inexpensively utilizedat low pressures to improve overall quality and safety of dairyproducts, as well as other liquid products, juices, extracts, liquidsupplements, and liquid pharmaceuticals. The combination ofrefrigeration below 7° C. and application of CO₂ pressure may result ina synergistic effect.

As previously stated, milk and dairy products are generally very rich innutrients that provide an ideal growth environment for manymicroorganisms. A principal class of microorganism that may find its wayinto milk is bacteria. Bacterial growth generally proceeds through aseries of four phases: (1) a lag phase during which time themicroorganisms become accustomed to their new environment with little orno growth; (2) a log phase during which bacterial logarithmic orexponential growth begins; (3) a stationary phase where the rate ofmultiplication slows down due to the lack of nutrients and build up oftoxins; and (4) a death phase in which bacteria numbers decrease asgrowth stops and existing cells die off. In addition, fungi such asyeast and molds, as well as bacterial viruses may also be present inmilk and dairy products. Typically, microbial growth will vary accordingto a number of factors including nutrient content, moisture content, pH,available oxygen, and temperature.

The 2003 Revisions of the Grade “A” Pasteurized Milk Ordinancepromulgated by the U.S. Food and Drug Administration establish chemical,physical, bacteriological and temperature standards for Grade “A” RawMilk and Milk Products for Pasteurization, Ultra Pasteurization orAseptic Processing. Principal among these are that milk be cooled to 10°C. (50° F.) or less within four hours or less of the commencement of thefirst milking and to 7° C. (45° F.) or less within two hours after thecompletion of milking provided that the blend temperature after thefirst milking and subsequent milkings does not exceed 10° C. Bacteriallimitations provide that the individual producer milk is not to exceed100,000 cfu per mL prior to commingling with other producer milk and notto exceed 300,000 mL as commingled milk prior to pasteurization.Bacterial counts are performed according to the Standard Plate Count(SPC) which determines the number of visible cfu's or colony-formingunits (numbers of individual or tightly associated clumps of bacteria)in 1 mL of milk incubated at 32° C. (90° F.) for 48 hours.

Milk is an excellent food source for humans, bacteria, andmicroorganisms alike as it is full of vitamins, fats, minerals,nutrients, and carbohydrates. Milk is rich in the protein casein whichgives milk its characteristic white color, and the most abundantcarbohydrate is the disaccharide lactose “milk sugar.” At roomtemperature, milk undergoes natural souring caused by lactic acidproduced from the fermentation of lactose by fermentive lactic acidbacteria. Spoilage is a term used to describe the deterioration of afood's texture, color, odor, or flavor to the point that it isunappetizing or unsuitable for human or animal consumption. Microbialspoilage of food often involves the degradation of protein,carbohydrates, and fats by microorganisms or their enzymes.

Several authors have reported on the use of CO₂ as an antimicrobialagent in foods including dairy products (Dixon and Kell, 1989; Haas etal., 1989). In raw milk, bacterial growth was reduced by 50% afteraddition of CO₂ and storage at 6.7° C. for 48 h (Shipe et al., 1978).King & Mabbitt (1982) demonstrated an extension in storage life of bothpoor and good quality milks by the addition of 30 ppm CO₂. CO₂ iseffective in reducing the rate of growth of organisms detected inaerobic plate count assays (Roberts and Torrey, 1988). Compared tocontrol milk, the SPC of milk containing 20-30 pmm dissolved CO₂ was 3log₁₀ cfu/ml lower after 4 days of storage at 7° C. (Mabbitt, 1982). Inthe presence of CO₂, the time for SPC to reach 7 log₁₀ cfu/ml wasextended from 3 to 9 days at 7° C. and 6 to 11 days at 4° C., whereas inthe control this level was reached in just 5 days at 7° C. and 8 days at4° C. (Hotchkiss, 1996). Coliforms and psychrotrophs were alsosignificantly reduced compared to control milk under the same conditions(Roberts and Torrey, 1988). Generally, gram-negative psychrotrophs aremore susceptible to the effects of CO₂, whereas gram-positive bacteriaand spores are more resistant; Lactobacillus spp. are relatively CO₂resistant, or their growth may be enhanced by a CO₂ enriched environment(Hendricks and Hotchkiss, 1997). Excessive growth of Lactobacillus spp.in raw milk may lead to spoilage or development of off-flavors due tofermentation. Treatments that reduce microbial populations may result inoutgrowth of thermoduric spore-forming bacteria due to reducedcompetition, increasing the likelihood of post-pasteurization spoilageor reduced food safety.

The addition of CO₂ has been shown to increase the lag phase of growthand decrease the growth rate of microorganisms (Martin et al., 2003). InCO₂-treated milk, extension of the lag phase increased the generationtimes of the Pseudomonas species (Roberts and Torrey, 1988). Increasingconcentrations of CO₂ increased lag phases and extended growth rates.King and Mabbitt (1982) demonstrated an extension in storage life ofpoor quality milk (10⁵ cfu/ml) by 1.2 days and good quality milk (10³cfu/ml) by 3 days with the addition of 30 ppm CO₂. The extension ofkeeping quality of milk due to CO₂ was maximized when the initial countsin the milk were low. Low-level carbonation of bulk tank milk inhibitsthe increase in microbiota for 3 to 4 days. The reduction in countswould, in turn, reduce the thermotolerant lipases and proteases secretedinto the milk, post-pasteurization (Espie and Madden, 1997).

Several theories explaining the mechanism of CO₂ action onmicroorganisms have been proposed. The exclusion of oxygen byreplacement with CO₂ may contribute to the overall effect by slowing thegrowth rate of aerobic bacteria (Daniels et al., 1985). CO₂ can alsoreadily pass through cell membranes, form carbonic acid within the cellwith a resultant decrease in intracellular pH which slows intracellularenzyme activities (Wolfe, 1980). CO₂ has been demonstrated to beinhibitory of certain enzymes, especially decarboxylating enzymes (Gilland Tan, 1979). Carbon dioxide can also accumulate in membrane lipidbilayers, altering membrane properties and inhibiting membrane functions(Enfors and Molin, 1978). The effect of CO₂ has been found to beenhanced at lower temperatures (Gill and Tan, 1979). The increasingsolubility of CO₂ at lower temperatures increased the relativeinhibitory effect of CO₂ on P. fragi (Enfors and Molin, 1981).

These studies have all addressed the use of CO₂ injections oratmospheres without subjecting the treated liquid to pressure. To someextent this may be due to the paucity of food grade pressure bulkstorage vessels. However, because the application of pressure to milk isknown to lead to undesirable biological changes, specifically theprecipitation of proteins, research has taught away from the presentinvention. When pressure has been applied to other perishable liquids,it has typically been at high pressures to achieve the substantialelimination of pathogens. The maintenance of milk and other bulkperishable liquids under low pressures of CO₂ at about 138 kPa and 350kPa for the purposes of retarding microbial growth during storage andtransport is heretofore unknown to the inventers.

While details of the carbon dioxide treatment according to the inventionmay be slightly varied according to the particular organic liquidsinvolved, the invention will be explained below in connection with milkcollection and processing, which may be best understood with referenceto FIG. 1.

FIG. 1 illustrates the movement of raw milk from a large or small dairyfarm 11,12 to a milk bottling or processing facility 15. Dairy farmsvary in size from a few cows to as many as 12,000 or more. The farmsmilk at least twice daily in milking parlors 21 and the milk is chilledand pumped into on-farm storage tanks 13. Milk is picked up by transporttankers 22 at least every other day and in the case of large farms 12,multiple times a day. Milk from small farms 11 is often taken toconsolidation facilities 14 where it is tested before commingling withother farms' milk. The process of this invention is to station carbondioxide injection systems 16 comprising tanks of liquefied or compressedcarbon dioxide with the appropriate hoses, regulators, valves, fittings,injectors and appurtenances necessary to dissolve the CO₂ into the milkat the farm 12 or consolidation facility 14 or in some cases the carbondioxide system 16 may be mounted on the transport vehicle so as to bemobile and not require separate systems 16 for each point of loading.

Once the raw milk is sufficiently infused with carbon dioxide forstabilization, the transport tank, a bulk pressure transport vessel 17such as intermodal tank 30 shown in FIG. 3, is pressurized to preservethe appropriate concentration of dissolved CO₂. The tank 30 can bepressurized using the same carbon dioxide source as for the infusion bythen rerouting the gas through the air inlet 33 as shown in FIG. 7.Alternatively, solid carbon dioxide, commonly referred to as “dry ice,”can be inserted into the liquid through the manway 35 shown in FIG. 3,at amounts calculated to create the required pressure when vaporized inthe sealed tank. This approach also has the added benefit of loweringthe temperature of the product, further slowing the growth of pathogensand enhancing the effectiveness of the invention.

The loaded tank 30 is transported to a milk bottling plant or processingplant 15 where the milk is agitated 18 to remove the carbon dioxide.This may be done in-situ by air injection into the transport tank or maybe done in a separate agitation tank to remove CO₂ to acceptable levelsor this may involve the use of vacuum or negative pressure. Theunloading process is otherwise essentially the same as with untreatedmilk except for this step.

The milk is then processed according to its end use 19 which may be topasteurize, homogenize, process, condense, culture, or perform othercustomary processes before packaging for retail sale or preparing forfurther transport or sale. Even in the event that the milk processingplant 15 is reasonably close to the dairy farm 11,12, there may still bea need to extend the life of the raw milk. The dairy farm or purchaserof the milk may want greater flexibility in processing the milk whensupply and demand are imbalanced. For instance, rather than divertingexcess milk to an alternative use, such as milk powder, a lower valueproduct, the milk processor may want to retain inventory at or near theplant 15 in times of greater supply for use later in times of greaterdemand. This preserves the milk for its highest and best use andeliminates unnecessary transportation cost. In the event it is desiredto transport the raw milk long distances, it is necessary today to userelatively expensive express truck delivery from farm 12 orconsolidation facility 14 to processing plant 15 as lower costalternatives are typically too slow. Even with express truck delivery,it is generally impractical to transport raw milk long distances withinthe three days desired or stipulated by industry or regulatory agenciesdue to the perishable nature of milk. With the greater shelf life of rawmilk stabilized according to the present invention, raw milk may beloaded at a dairy farm with a carbon dioxide mixture and sealed withpressurized carbon dioxide and the tank delivered to a rail carrier forconveyance anywhere in North America or to an ocean carrier forconveyance to much of the world.

FIG. 2 shows the process that has been utilized to supply milk toHawaii, a variation of the milk distribution system described in FIG. 1.Here, raw milk is collected from farms 11, 12 by transport tankers 22and delivered to milk processing facilities 15 near the ports 27 inCalifornia using traditional transport means. At the processingfacilities 15, the raw milk is pasteurized 23, chilled and pumped intotank containers 24. The tank containers are taken to the shipping docks27 at the port and transported by cargo ship to Hawaii. In Hawaii, thetank container is discharged from the ship and delivered to another milkprocessing facility 28 which re-pasteurizes 23 the milk prior topackaging 25 for retail. Alternatively, packaged milk 25 in SouthernCalifornia may be placed in refrigerated containers 26 and transportedby cargo ship 27 to Hawaii for delivery to the customer 29. Thisalternative eliminates the necessity of pasteurizing the milk twice, butincurs the additional expense of refrigerated shipment and eachprepackaged unit has fewer days remaining on its shelf life whendelivered to Hawaiian retailers than it would if the milk were processedlocally. According to the present invention, these problems are solvedby placing raw milk in pressured tank containers 17 with carbon dioxide16, pressurized, and shipped 27 without either pre-pasteurization orrefrigeration.

A preferred transportation container for use in practicing the inventionis a vessel sufficiently large so as to hold maximum legal highwayweights of product (in the United States roughly 50,000 pounds) and of asufficient volume to allow the head space to be pressurized. Dependingupon the design and tare weight of the pressure vessel and upon thespecific gravity of the product hauled, such a unit is preferablybetween about 4500 and 6500 U.S. gallons in capacity. The containershould be food grade or sanitary grade depending upon the producthauled, insulated against significant temperature gain or loss, built asa pressure vessel with a bottom discharge outlet, pressure/vacuum reliefvalve, and an air inlet. To meet pressure vessel codes and be foodgrade, construction of a good grade of stainless steel such as 304 or316 is most typical, but manufacture from other metals such as titaniumor of a composite material such as carbon fiber is also possible. Thepreferred insulating material is a cellular foam, and it is desirablethat the insulation provide the container with an R-value of at leastabout 27.5 and preferably between about 28 and 36. A particularlypreferred container is a super-insulated food grade tank container 30,typified by the 22,000 liter model HO4 tank utilized by Agmark Foods,Inc., as shown in FIG. 3.

As shown in FIG. 3, the food grade tank container 30 is constructed as acylindrical pressure vessel 37, mounted within frame 31 to enable thecontainer to be shipped by truck, rail or ocean. However, suitable tanksmay be built for dedicated truck use or as railcars in carload serviceor adapted to other modes of transportation including bulk oceanshipments. The tank 30 in FIG. 3 has a pressure relief valve 32, an airinlet 33, and a bottom discharge outlet 34. In addition, a manway 35 islocated at the top of the tank.

A preferred method of loading a transport container 30 as shown in FIG.3 according to the present invention is to pump the milk with a foodgrade pump 40, shown in FIG. 6, the pump 40 being either on-farm ortruck mounted, from its on-farm storage container 13 or by air pressureapplied to the on-farm tank 13 (if it is a pressure vessel) or by vacuumapplied to the tank container 13, if so designed. The milk will flowthrough the hoses 41 from the storage tank 13 to an inlet of thetransport container 30, generally the bottom discharge assembly 34 shownin FIG. 3. If the product has not already been treated with carbondioxide, the carbon dioxide will be dissolved into the milk 49 by use ofa sparge or fritted nozzle 48, as illustrated in FIG. 4. The nozzlebreaks the gas into microscopic bubbles that are easily dissolved intothe liquid while the liquid is under some amount of back pressurebetween the storage tank 13 and the transport tank 17. The amount ofcarbon dioxide applied is regulated by traditional gas regulators 46between the carbon dioxide source 45 and the fritted nozzle 48 so thatthe absorption is achieved at the rate appropriate for the liquidinvolved. For milk, a CO₂ concentration of between about 200 and 2000parts per million is desired, although levels at 2400 ppm may berealized with satisfactory results.

Unloading is accomplished by attaching a hose or stainless steel pipe tothe discharge valve 34 of the tank container 30, and to a pump 40. Thepump empties the contents of the transport tank 30 through the hose 41into a plant 15 storage tank for use in the plant's system. According tothis invention, it is also possible to unload milk or other organicliquids without use of a pump. This is accomplished by attaching acompressed purified air system such as from triple filtered system 50 inFIG. 8 to the air inlet 33 on the tank 30 and using both the originalCO₂ pressure and purified air to push the product out of the tank. Inthe case of milk, it is widely understood that pumping is both necessaryand undesirable; necessary in that current milk transport equipment doesnot accommodate pressure and undesirable in that pumping has a tendencyto shear fat molecules in a way that can encourage rancidity. Theinvention facilitates a completely new set of business practices thatcan dramatically improve the quality, price, and service associated withmoving perishable organic liquids.

EXAMPLES

The first example is a laboratory scale experiment to investigate theeffect on raw milk spoilage and pathogenic microbia of holding raw milkunder positive CO₂ pressures that do not result in precipitation of milksolids. Changes in total Lactobacillus spp., lactose fermenting andnon-lactose fermenting gram-negative bacteria, Escherichia coli,thermoduric bacteria and SPC were examined as indicia of potential milkquality and safety.

Test System Design

The apparatus for pressurizing and holding raw milk samples is shown inschematic form in FIG. 9 and consisted of two 13-ml stainless steel1.27-cm OD cylindrical vessels 60, 61, one vessel 60 was pressurizedwhile the other served as a control 61. Compressed and filtered CO₂ froma high-pressure tank 62 was used (Empire Airgas, Inc, Elmira, N.Y.). Thesystem consisted of pressure regulator 63, a fine metering valve 64(NUPRO Company, Willoughby, Ohio), an on-off valve 65 (Circle Seal,Anaheim, Calif.) and a check valve 66 (NUPRO Company, Willoughby, Ohio).The fine metering valve controlled gas flow such that the time to reachdesired pressure was less than five seconds. The gas entered thevertically positioned treatment vessel 60 from the bottom and was thusbubbled through the milk until the set pressure was reached. A checkvalve 66 was placed immediately before the inlet to the pressure vesselto prevent the backward flow of the fluid milk into the gas inlet line.The outlet of the vessels consisted of a pressure gauge 67 and ahigh-pressure release valve 68 (High Pressure Equipment, Erie, Pa.). Therelease valve 68 was kept tightly closed during treatment. The controlvessel 61 was closed off from both ends but not connected to the carbondioxide line inlet and outlet lines.

The apparatus was cleaned and sanitized before and after each treatmentas follows: water rinse, Conquest sodium hydroxide (Ecolab Inc., St.Paul Minn.) soak (20 min, 23° C.), warm tap water rinse (50° C.),Monarch CIP phosphoric acid bath immersion (Ecolab Inc., St. Paul,Minn.) (20 minute, 23° C.), warm tap water rinse (50° C.);Tricholoro-o-cide XP (Ecolab Inc, St. Paul, Minn.) soak (30 min, 23°C.), sterile water (50° C.) rinse (3×). This protocol was validated bytesting swab samples of critical control points in the dismantledapparatus for microbial load, and testing equipment rinse water pH andresidual chlorine content (Hach Company, Loveland, Colo.). Temperaturewas controlled by a circulating water bath 70 (VWR 1145 RefrigeratedTemperature Constant Circulator), which circulated hot/cold waterthrough copper coils 71, immersed into water in a vacuum dewar flaskthat held the treatment and control vessels. Copper-Constantanthermocouples 72 measured the temperature of the treatment and controlvessels, and were continuously logged onto a temperature recorder 73(Omega Engineering Inc, Stamford, Conn.).

Milk Samples and Treatments

Whole, unhomogenized, raw milk was obtained from two sources. Commingledmilk samples were obtained from the Northeast Dairy Herd ImprovementAssociation, Inc. (Ithaca, N.Y.), a dairy analytical consultinglaboratory. These samples were commingled bulk milks from 236 farms fromNew York, Pennsylvania, and New Jersey and thus, could be consideredrepresentative of a wide range of milk flora. Milk was also obtainedfrom the Cornell University Teaching and Research Center bovine herd(T&R Center; Dryden, N.Y.). All milk was stored at 6° C. until use. Rawmilk from the T&R Center was received in less than 12 hours aftermilking in sterile bottles and held on ice until it could be moved to a6° C. cooler.

Milk samples were mixed and 5 ml of milk added into the treatment andcontrol vessels. The treatment vessel was connected to the apparatus andthe control vessel closed off. Both vessels were placed in the waterbath. When the desired temperatures were attained in both treatment andcontrol vessels, CO₂ was introduced through the bottom of the treatmentvessel until the set pressure was reached. The CO₂ pressure wasmaintained throughout the test period. When the desired time wasreached, the CO₂ inlet was turned off, the pressure release valve on theoutlet line opened, and the pressure released in under one minute. Afterdepressurization, the treatment and control vessels were removed fromthe water bath and their external surfaces were wiped dry, sanitizedwith 95% ethanol, detached from the apparatus and transferred intosterile containers for dilution and plating.

The effect of CO₂ pressures and temperature combinations on proteinprecipitation was measured at CO₂ pressures of 344, 689, 1378, 2067,2757, and 3446 kPa at 20, 10, and 5° C. for 5, 15, 30 and 60 min. Theamount of protein precipitation was quantified and expressed aspercentage precipitated solids by the method of Tomasula (1995).

Short (<1 h) and longer term (1, 4 and 9 day) experiments wereconducted. Raw milk (Northeast Dairy Herd Improvement Association, Inc.;Ithaca, N.Y.) in 5 ml aliquots was treated at each of the followingcombinations of CO₂ pressure, temperature, and time (kPa/° C./min):1378/5/15, 2757/5/5, 3446/5/5. In longer term studies, raw milk from theT&R Center was first stored at 6° C./48 h so that the SPC were atdetectable levels at treatment initiation. Five ml of milk were treatedwith CO₂ pressures of 0 (control), 68, 172, 344, 516 and 689 kPa for 1to 9 days at 4.1 to 10° C.

Raw milk from the T&R Center was monitored for changes in aerobicbacteria, gram-negative bacteria and total Lactobacillus spp. asfollows: CO₂ pressures of 0 (control), 68, 172, 344, 516 and 689 kPa, at6.1° C. for 4-days. SPC, gram-negative bacteria and total Lactobacillusspp. were enumerated before (day 0) and after (day 4) treatment. Gramnegative bacteria were enumerated on MacConkey Agar (Difco Manual,Becton Dickinson & Co., Sparks, Md.), a selective and differential mediawhich can be used to discriminate between lactose fermenting andnon-lactose fermenting gram-negative bacteria. Use of this media allowsa one-step method of obtaining estimates of both coliform andnon-coliform gram negative bacteria. Coliforn bacteria may includespecies of Escherichia, Klebsiella and Enterobacter, potential pathogensand/or spoilage organisms. Non-coliform gram negative bacteria mayinclude spoilage organisms such as pseudomonads or potential pathogenssuch as Salmonella spp. or Shigella spp. Numbers of Lactobacillus spp.populations were estimated using acidified (adjusted to pH 5.5 withglacial acetic acid) Lactobacillus MRS agar (Difco Manual, BectonDickinson & Co., Sparks, Md.) after incubation under anaerobicconditions; suspect colonies were confirmed by gram stain.

The time to reach an SPC of 2×10⁵ cfu/ml was determined using raw milk(T&R Center) without a 2-day storage time. Equal volumes weretransferred into treatment and control vessels and held at 0 and 689 kPaCO₂ and 4.1° C.

The progression of these counts (total, Coliform/E. coli and thermoduricbacteria) in the treatment and control samples was tracked by conductingchecks on the total aerobic counts (SPC) on treatment days 4 and 6.Based on the levels of total counts on days 4 and 6, analyses of totalcoli forms/E. coli and thermoduric bacteria after day 6 were conductedeither in 1-day or 2-day intervals. The control sample final count wasmeasured on days 4 and 6.

Microbiological Methods

For all microbiological assays, milk sample aliquots of 1 ml were usedin dilution series. Standard Plate Counts (SPC) were performed by themethod described in Standard Methods for the Examination of DairyProducts (Houghtby et al., 1992). Gram-negative bacteria were enumeratedon MacConkey agar (Difco Manual, Becton Dickinson & Co., Sparks, Md.)after spread plating and incubation at 30° C. for 48 h. This selective,differential media was used to estimate total lactose fermenting,non-lactose fermenting and total gram negative bacteria. Lactobacillusspp. were estimated by pour plating in acidified Lactobacillus MRS agar(Difco Manual, Becton Dickinson & Co., Sparks, Md.), incubated at 32° C.for 48 hours under anaerobic conditions. Representative and distinctivesuspect colonies were gram stained, and confirmed gram positive bacillicolonies were counted as an estimate of total Lactobacillus spp.

Initial total, coliform, and thermoduric counts were each determined forcontrol and treated samples. Thermoduric organisms were enumerated bythe laboratory pasteurization count (LPC) method described in theStandard Methods for the Examination of Dairy Products (Houghtby et al.,1992). The 3M Petri film count plate (3M Microbiology Products, St.Paul, Minn.) was used to enumerate total coli forms and Escherichia coliin the raw, treated and control milk samples.

Statistical Methods

MINITAB Release 13.1 (Minitac Inc, State College, Pa.) was used forstatistical analyses of the data. Analysis of Variance (ANOV A) was usedto determine the effect of CO₂ pressure, and the interaction effects ofpressure and temperature.

Results

Application of CO₂ pressures greater than 1378 kPa (200 psi) for 15 to60 min resulted in more than 1% precipitation of milk solids at 20° C.(data not shown). Treatment for 30 min at 2067 kPa (300 psi) resulted in2.6% (w/w) solids which approached the maximum (2.8%) found by sulfuricacid precipitation (Southward, 1986); However, lowering the holdingtemperature reduced the amount of precipitation; at 5° C. and pressuresof less than 2067 kPa precipitation could not be detected, even after 60min. Treatment combinations of 689 kPa for 60 min, 1378 kPa for 30 min,2757 kPa for 5 min and 3446 kPa for 5 min did not cause detectableprecipitation at 5° C.

These results generally agree with previous reports including Jordan etal. (1987), Tomasula (1995), and Calvo and Bacones (2001), whoindependently investigated the precipitation of caseins from raw skimmilk using pressurized CO₂. Tomasula (1995) found that CO₂ pressuresbetween 2757 and 5514 kPa and temperatures between 38 and 49° C. causedcomplete casein precipitation. Calvo and Bacones (2001) precipitated 85%of raw skim milk caseins by applying CO₂ pressures above 1998 kPa for 3h at 40° C. Jordan et al. (1987) obtained 99% precipitation of skim milkcasein by treatment with 3515 kPa at 50° C.

Protein precipitation occurs when the pH of the milk has been reducedbelow the isoelectric point of the casein (pH 4.6). The addition of CO₂to milk leads to the formation of carbonic acid and a decrease in pH. Inaddition, pressurization with CO₂ can cause precipitation of caseins ata pH higher than its isoelectric point (Tomasula et al., 1999). Ma andBarbano (2003) found that increasing CO₂ concentration and pressuredecreased the pH of skim milk; the pressure effect was greater as CO₂concentrations increased. These researchers also determined thatincreasing temperature affected the solubility of milk colloidal calciumphosphate, resulting in a decrease in milk pH. Jordan et al. (1987)found that precipitation of casein occurred between 40 and 70° C., andthat the yield at any specific temperature was dependent upon a minimumpressure; this minimum pressure was inversely related to temperature.Thus, specific pressure/time/temperature treatment combinations must bemanipulated so that the conditions do not cause precipitation ofproteins from raw milk.

All time-pressure combinations significantly reduced the SPC of the rawmilk compared to untreated controls, even at a low pressure and hightemperature combination of 68 kPa and 20° C. At 1378 kPa, the controlSPC was 7.89 log₁₀ cfu/ml while the treated milk SPC was reduced by 0.33log₁₀ after 15 min and 0.39 log₁₀ after 30 min. Twenty-four hourtreatments at 20° C. and pressures ≧344 kPa resulted in microbialinactivation. The SPC of milk treated at 344, 516 and 689 kPa wassignificantly reduced from initial SPC by 0.39, 0.62 and 0.82 log₁₀,respectively, while the SPC of the control milks significantly (P<0.05)increased by as much as 2.06 log₁₀ cfu/ml. SPC in milk held at 68 and172 kPa significantly increased by 1.07 and 0.59 log₁₀ cfu/ml,respectively, however this population increase was significantly lessthan that exhibited by the control milk.

Carbon dioxide pressure treatments of 68 and 172 kPa at 10° C. appliedover 24 h were more effective at curtailing growth than similarpressure-time treatments at 20° C. As found at 20° C., there was a lossin viability at pressures ≧344 kPa and the differences between controland test counts increased with increasing holding time; significantdecreases in counts of 0.31, 0.56 and 0.71 log₁₀ cfu/ml at 344, 516 and689 kPa CO₂, respectively, were achieved. The difference in SPC betweencontrol and test milks at 689 kPa was 2.68 log₁₀ cfu/ml. These dataindicate that holding raw milk under CO₂ pressure not only slowed thegrowth of the microorganisms in the raw milk but in some cases alsosurprisingly resulted in a loss in viability of the microorganisms atrelatively low pressure levels of only 344 kPa.

The pH of the treated and control milk samples (as measured atatmospheric pressure) was 6.6 to 5.9 at CO₂ pressures ≦516 kPa and 5.7when treated at pressures ≧516 kPa and 20° C. The pH of the treated andcontrol milk samples, when treated at 10° C., was 5.5 at CO₂ pressures≦516 kPa and 5.8 when treated at pressures ≧516 kPa.

Others have shown inactivation of microbiota in raw and pasteurized milkwith CO₂ at significantly higher pressures (Erkman 1997 and 2000; Calvoand Bacones, 2001). Calvo and Bacones (2001) found a decrease in bulkraw milk microbiota of 2 log₁₀ cfu/ml upon treatment with 3997 kPa (or5800 psi) CO₂ at temperatures ≧40° C. for 30 min. Erkman (2000)demonstrated a reduction in aerobic microorganisms in whole milk of 6log₁₀ cfu/ml after a 24 h treatment under 6044 kPa CO₂ pressure at 45°C. Erkman (1997) also demonstrated a reduction of 8 log₁₀ cfu/ml after a5-h 14598 kPa CO₂ treatment at 25° C. However, the use of these CO₂pressures would in our experience result in complete precipitation ofthe caseins and would require the use of specially designed equipment.Calvo and Bacones (2001) reported that pressures of 3997 kPa causedprecipitation while Erkman (1997 and 2000) made no mention of the stateof the milk. These high pressures, over 3000 kPa, are more appropriateas substitutes for thermal pasteurization of liquids that do not sufferfrom protein precipitation, but due to equipment requirements are notgenerally suitable for bulk storage and transportation purposes.

Lowering the holding temperature to 6.1° C. significantly reducedmicrobial growth compared to control milks when CO₂ pressures of 68,172, 344, 516 and 689 kPa were applied for 4 days. For example, the SPCof milk held at 689 kPa was 0.89 log₁₀ cfu/ml lower than initial countsand 3.48 log₁₀ cfu/ml lower than the controls. Over the course of 9days, storage under 689 kPa CO₂ at 4° C., the ratios of treated tountreated SPC, thermoduric, coliform, and E. coli counts wereconsistently lower than the ratios of control to untreated counts forthe comparable groups (cfu/ml) as summarized below in Table 1. TABLE 1Counts, cfu/ml Untreated, Treated Control Day 0 4 Days 6 Days 8 Days 9Days 4 Days 6 Days 8 Days 9 Days SPC 3.0 * 10^(3a) 7.8 * 10^(2b) 4.4 *10^(4c) 9.7 * 10^(4d) 2.4 * 10^(5e) 1.4 * 10^(5e) 1.2 * 10^(6f) 7.3 *10^(6g) 9.7 * 10^(6h) Thermoduric 1.0 * 10⁰ 1.0 * 10⁰ 9.0 * 10^(0m)2.1 * 10^(1n) 3.7 * 10^(1o) 1.0 * 10⁰ 8.1 * 10^(1p) 9.6 * 10^(1q) 1.0 *10^(2r) Bacteria Coliforms 1.1 * 10^(2h) 1.0 * 10^(2h) 1.0 * 10^(2h)9.3 * 10^(1h) 9.0 * 10^(1h) 4.0 * 10^(2h) 4.3 * 10^(2i) 5.5 * 10^(2i)7.8 * 10^(2j) E. coli 2.0 * 10^(1k) 1.7 * 10^(1k) 1.7 * 10^(1k) 1.7 *10^(1k) 1.5 * 10^(1k) 3.6 * 10^(1l) 3.9 * 10^(1l) 6.1 * 10^(1l) 7.4 *10^(1l)The effect of 689 kPa CO₂ pressure at 4° C. after 4, 6, 8 and 9-daytreatments on the SPC, thermoduric bacteria, coliforms and E-coli countsin untreated, treated and control raw milks. Experiment conducted induplicate, n = 2 (2 milk samples analyzed), each sample plated intriplicate. Counts with different letters are significantly different (P≦ 0.05).

Milks treated at 68, 172, 344 and 516 kPa significantly increased froman initial SPC of approximately 3.30 log₁₀ cfu/ml by 1.28, 1.10, 0.94and 0.82 log₁₀ cfu/ml, respectively, while the control SPC increased by2.86, 2.85, 2.86 and 2.93 log₁₀ cfu/ml, respectively. Milk held at 689kPa treatment at 6.1° C. for 4 days exhibited greater inactivation thanthat exhibited after the 10 or 20° C. 24 h treatments (P<0.05). The pHdecreased from 6.6 before treatment to 5.5 in milks treated at 516 kPa,5.8 at 344 kPa and 5.9 at 68 kPa.

In addition to SPC, there were significant differences in gram-negativelactose fermenting and non-lactose fermenting bacteria and Lactobacillusspp. between CO₂ treated and control milks as shown in FIGS. 10 a-10 e.In these bar charts, the first bar is gram-negative lactose fermentingbacteria, the second gram-negative non-lactose fermenting bacteria, thethird Lactobacillus spp.; and the final bar is SPC. Thus, levels ofgram-negative fermenters and non-fermenters were reduced at allpressures compared to untreated controls. Likewise, Lactobacillus spp.counts were approximately 1 to 2 log₁₀ cfu/ml lower in the test milkscompared to control milk. At 689 kPa, gram-negative lactose fermentingand non-lactose fermenting bacteria exhibited significant decreases of0.80 and 0.64 log₁₀ cfu/ml, respectively, compared to initial counts.Under 516 kPa CO₂ pressure, SPC of treated samples were notsignificantly different from initial untreated samples while SPC ofcontrol samples increased by 2.95 log₁₀ cfu/ml Reductions in totalmicrobial populations as well as reductions in gram-negative andLactobacillus spp. populations would result in improved quality of theraw milk. Ruas-Madiedo et al. (1996) found that lower levels of volatilecompounds (ethanol, 2-propanone, and 2-butanone, which are microbialmetabolites) were produced in carbonated milk during storage and thathigher sensory scores were achieved than in untreated milks. In a laterstudy, Ruas-Madiedo et al. (2000) found a direct association betweenreduced microbial growth and reduced levels of microbial glucosidases inraw milk stored with CO₂; degradation of milk glucose was subsequentlyreduced in the treated milks. It has also been found that levels offat-soluble vitamins (retinol, -β-carotene and α-tocopherol) in milktreated with CO₂ and stored at 4° C. for 7 days were higher than thatmeasured for untreated raw and pasteurized milks (Ruas-Madiedo et al.,1998a, b).

In the current study, populations of Lactobacillus decreased after CO₂pressure treatment. Others have found that treatment with CO₂concentrations between 0 and 2000 mg/l had no impact on the lag phase ofLactobacillus sake when grown at 7° C., and influences on the maximumspecific growth rate was least affected as compared to species ofPseudomonas, Aeromonas, Bacillus, Brochothrix and Shewanella(Devlieghere and Debevere, 2000). Espie and Madden (1997) reported noeffect of 30 and 45 ppm CO₂ on the growth of Lactobacillus spp. Neitherof these investigations, however, incorporated pressures aboveatmospheric in their treatments. Reductions in populations ofLactobacillus plantarum of more than 6 logs was achieved after treatmentwith CO₂ pressures of 13 MPa at 30° C. for 30 minutes (Hong and Pyun,1999). In subsequent studies, these researchers found that high pressureCO₂ treatment of L. plantarum resulted in irreversible cellular membranedamage and reduced activity of some intracellular enzymes, physiologicalchanges that could result in microbial inactivation (Hong and Pyun,2000). Combined or enhanced effects of low pressures and CO₂ treatmentscould explain the observed reductions in total Lactobacilluspopulations.

The effect of 689 kPa CO₂ at 4° C. on the time to reach an SPC of 10⁵cfu/ml was investigated. Pasteurized Milk Ordinance Grade A regulationsspecifies the SPC for raw milk should be less than 10 ⁵ cfu/ml prior topasteurization. As shown in FIG. 11, where the columns sequentiallyrepresent total counts, thermoduric bacteria, total coliforms, and E.coli, the treated milks reached 10⁵ cfu/ml after 8 days of treatment,whereas the control milk reached this level after just four days.Treatment at 689 kPa and 4° C. extended the treatment holding time atleast four days as compared to the control. At the end of four daystreatment, treated milk SPC had decreased to 2.89 log₁₀ cfu/ml from 3.48log₁₀ cfu/ml while control milk SPC increased by nearly 5 log₁₀ cfu/ml.This reduction in SPC in treated milk agrees with the trend observed inthe four-day experiments conducted at 6.1° C. (FIG. 10). Milk SPCincreased to 4.64, 4.99 and 5.37 log₁₀ cfu/ml after 6, 8 and 9 daystreatment, respectively (FIG. 11). Neither E. coli nor total thermoduricbacteria counts increased in the treated milk but both significantlyincreased in the controls. The pH of the treated milk samples changedfrom an initial value of 6.6 to 5.5 at the end of days 4, 6, 8 and 9 oftreatment.

Example 2

This second example was an experiment designed to confirm thepreliminary results of the first example on a commercial or bulk scale.

Test System Design

The apparatus pressurizing and holding raw milk samples consisted of a5300 U.S. gallon (20,000 liter) food grade, insulated, pressure vesselshipping container of the model HO4 type utilized by Agmark Foods, Inc.Compressed and filtered CO₂ from high pressure tank was used, includinga Praxair inline 3A sparger. The Agmark shipping container was sanitizedon Aug. 4, 2004. The following day, the tank was pre-chilled with aspray of CO₂ and filled with 3,291 U.S. gallons of fresh raw milkobtained from the Cornell University Teaching and Research Center BovineHeard (T&R Center; Dryden, N.Y.) on Aug. 5, 2004. The raw milk from theT&R Center was received less than 12 hours after milking and wasintroduced from a holding tank into the pressure vessel at a temperatureof approximately 1 to 2° C. Prior to filling the pressure vessel, thevessel was sealed and pressurized with carbon dioxide to a pressure of25 psi (172.5 kPa). Milk was pumped into the tank through the dischargevalve at the rear of the tank and CO₂ was injected into the flow of themilk with an inline Praxair 3A sparger at a rate sufficient to infuseapproximately 2000 parts per million at a gas flow rate of approximately16 cubic feet per minute. The raw milk was pumped into the vessel at aflow rate of 70 to 80 gallons per minute with flowing CO₂ under a 40 psiline pressure and 25 psi tank pressure. At the conclusion of loading,the temperature of the milk was at 2.5° C. with a tank pressure of 43psi.

Samples were taken daily from 17 consecutive days from both the top andbottom of the pressure vessel. A control sample of five gallons ofuntreated milk was held at approximately 2° C. for the duration of thestudy and similarly sampled. Microbiological methods consistent withthose described in the first example were utilized.

The data in Table 2 reflects bacterial growth (SPC log cfu/ml),thermoduric count psychrotropic count, E. coli and coliform count, pH,and CO₂ content, while Table 3 provides measurements of milk andenvironmental temperatures. TABLE 2 Laboratory test data for control andCO2 treated whole raw milk: SPC, Thermoduric count, Psychrotrophiccount, E. coli and Coliform count, Ph, CO2 content E. coli/ColiformsDate Day Control SPC Top cufu/ml Bottom Control Top Bottom 80504 13.69E+03 2.99E+03 3.19E+03  <1e0 9.15E+01  <1e0 8.95E+01  <1e0 1.10E+0280604 2 6.00E+03 2.04E+04 5.05E+02 <1e1 4.50E+01 1.00E+01 9.95E+021.00E+01 1.00E+01 80704 3 3.00E+03 4.30E+02 3.40E+02 1.00E+01 1.75E+02<1e1 <1e1 1.50E+00 <1e0 80804 4 3.95E+03 2.69E+02 1.02E+02 1.00E+003.00E+01 1.00E+00 4.00E+00 <1e0 1.00E+00 80904 5 3.95E+03 2.00E+021.05E+02 2.00E+00 3.15E+01 <1e0 5.00E+00 <1e0 <1e0 81004 6 2.75E+031.89E+03 1.01E+02 2.00E+00 2.95E+01 1.50E+00 1.85E+01 <1e0 <1e0 81104 72.84E+03 1.76E+03 3.25E+02 1.00E+00 2.90E+01 <1e0 1.15E+02 <1e0 1.45E+0181204 8 3.49E+03 1.61E+04 1.77E+02 <1e0 2.15E+01 <1e0 2.72E+02 <1e06.30E+01 81304 9 4.45E+03 1.27E+04 4.20E+01 <1e0 2.10E+01 <1e0 2.84E+02<1e0 <1e0 81404 10 3.65E+03 9.55E+03 4.50E+01 — — — — — — 81504 118.65E+04 3.14E+04 5.05E+01 <1e0 1.40E+01 <1e0 1.60E+02 <1e0 <1e0 8160412 1.76E+05 4.50E+04 2.05E+01 — — — — — — 81704 13 1.12E+06 4.10E+052.00E+01 <1e0 1.50E+01 <1e0 <1e0 <1e0 <1e0 81804 14 <1e0 9.15E+01 <1e08.95E+01 <1e0 1.10E+02 81904 15 <1e1 4.50E+01 1.00E+01 9.95E+02 1.00E+011.00E+01 82004 16 1.00E+01 1.75E+02 <1e1 <1e1 1.50E+00 <1e0 82104 171.00E+00 3.00E+01 1.00E+00 4.00E+00 <1e0 1.00E+00 82204 18 2.00E+003.15E+01 <1e0 5.00E+00 <1e0 <1e0 Ph ppm CO₂ Thermodurics PsychrotrophicsCon- Con- Date Control Top Bottom Control Top Bottom trol Top Bottomtrol Top Bottom 80504 3.15E+02 1.00E+01 1.00E+01 <1e1 <1e1 <1e1 6.6 5.95.9 129 2001 2190 80604 8.20E+02 4.25E+02 5.00E+01 6.50E+00 <1e0 <1e06.6 5.9 5.9 103 1988 2004 80704 5.10E+02 4.50E+02 3.50E+00 8.00E+005.00E+00 5.00E+00 6.7 6 5.9 103 2125 1985 80804 5.20E+02 6.00E+001.00E+00 2.10E+01 2.00E+00 3.00E+00 6.7 6 5.9 94 2091 1950 809045.20E+02 1.00E+00 1.00E+00 3.10E+01 <1e1 <1e1 6.8 6 6 90 2070 2071 810042.81E+02 5.00E+01 3.50E+00 6.8 6 5.9 90 2055 2128 81104 1.89E+024.00E+01 2.00E+00 6.8 6 5.9 90 2274 2043 81204 2.80E+02 2.22E+031.50E+00 6.7 6 6 90 2898 1970 81304 1.89E+02 1.15E+03 <1e0 6.8 6 6 902884 1920 81404 6.15E+02 6.45E+02 3.00E+00 6.8 6 6 90 2250 2146 815042.05E+02 5.20E+02 <1e0 6.7 6 6 90 2740 1943 81604 4.55E+02 8.05E+03 <1e06.8 6 6 90 2477 2060 81704 4.75E+02 1.77E+02 1.00E+00 6.8 6 6 100 22502146 81804 3.15E+02 1.00E+01 1.00E+01 6.8 6 6 103 2236 2126 819048.20E+02 4.25E+02 5.00E+01 6.7 6 6 103 3311 2198 82004 5.10E+02 4.50E+023.50E+00 82104 5.20E+02 6.00E+00 1.00E+00 82204 5.20E+02 1.00E+001.00E+00

TABLE 3 Temperature date (° F. in the vicinity of the Hartford, NYCornell University Teaching and Research Center (CLIMOD database) fromTompkins County, Ithica station Sample Sample Temperature ° C.Temperature ° F. High Low Avg Day Day Control Top Bottom Control TopBottom Temp Temp Temp 80504 1 2 2.6 2.6 36.6 36.6 36.6 75 61 68 80604 22 2.3 2.6 35.6 36 36.4 69 47 58 80704 3 2 4.6 2.7 35.6 40.3 36.7 62 5257 80804 4 2 4.1 2.9 35.6 39.4 37.3 65 53 59 80904 5 3 4.1 3.8 37.4 39.839 74 51 63 81004 6 2 7 4.9 35.6 44.7 40.5 80 57 69 81104 7 2 8.5 4.835.6 47 40.3 80 61 71 81204 8 2 8.9 5.6 35.6 47.6 41.9 75 60 68 81304 92 8.8 5.8 35.6 47.4 42.1 66 60 63 81404 10 2 10.1 6.4 35.6 49.5 43 72 5966 81504 11 2 10.1 8.9 35.6 50 47.7 73 53 63 81604 12 2 10.4 8.2 35.650.4 46.5 75 55 65 81704 13 2 10.4 8.4 35.6 50.6 47 74 50 62 81804 14 210.7 8.5 35.6 50.8 47.2 80 53 67 81904 15 2Comment: Bottom tank sample averaged a daily increase of 0.75 degree F.while the top tank sample averaged a daily increase of 1 degree F.

A microbial quality limit of 5 log cfu/ml SPC was adopted from the U.S.Department of Health and Human Services, Public Health Service and Foodand Drug Administration Grade A PMO standards for individual producergrade “A” raw milk. As shown in FIG. 12 a, the total microbial growthdid not reach levels of the quality limit until day 11 for the controlmilk and day 12 to 13 for the top tank milk. Bacterial levels in thebottom of the tank did not reach the limit during the entire 17 daystudy and actually decreased from the first day's measurement althoughthe temperature had increased to approximately 9° C. (FIG. 12 b) The lowpressure CO₂ storage extended the shelf life of the raw milk by 4 to 5days with no added refrigeration or measurable increase in food safetyrisk. Due to the stationary nature of the test, milk fat separated andformed a denser layer at the top in which most pathogens wereconcentrated. A natural agitation of raw milk product during transitwould mitigate this effect. Certain low fat products may actuallyachieve reduced microbial counts over time under similar CO₂ pressures.

REFERENCES

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All publication, patent, and patent documents are incorporated byreference herein as though individually incorporated by reference.Although preferred embodiments of the present invention have beendisclosed in detail herein, it will be understood that varioussubstitutions and modifications may be made to the disclosed embodimentdescribed herein without departing from the scope and spirit of thepresent invention as recited in the appended claims.

1. A method for extending the shelf life of perishable liquidscomprising the steps of mixing the liquid with carbon dioxide (CO₂),placing the CO₂ mixed liquid in a pressure vessel and applying andmaintaining a CO₂ head pressure of between about 138 kPa and 350 kPa. 2.The method of claim 1 wherein the CO₂ mixed liquid is introduced intothe vessel at a temperature of less than 10° C., and the vessel isinsulated.
 3. The method of claim 1 wherein the pressure vessel isrefrigerated, and the CO₂ mixed liquid is held in the pressure vessel ata temperature no greater than 7° C.
 4. The method of claim 1 wherein theliquid is milk.
 5. The method of claim 1 wherein the pressure vessel isan intermodal tank.
 6. The method of claim 4 wherein the Standard PlateCount of the liquid does not increase after four days in the pressurevessel.
 7. The method of claim 1 wherein the CO₂ head pressure ismaintained between 207 kPa and 350 kPa.
 8. A process to inhibit thegrowth of microorganisms in a perishable liquid comprising: (a) addingcarbon dioxide (CO₂) to the liquid; (b) placing the CO₂ enhanced liquidin a pressure vessel subject to CO₂ head pressure sufficient to inhibitgrowth of microorganisms without subjecting the liquid to undesirablebiological changes.
 9. The process of claim 8 wherein the liquid is rawmilk.
 10. The process of claim 8 wherein the liquid is maintained at atemperature less than 10° C.
 11. The process of claim 8 wherein theliquid includes at least one of the group consisting of milk fat, milkprotein, and whey.
 12. The method of claim 9 wherein the CO₂ is mixedwith the liquid and maintained at a pressure and temperature so that theresulting pH of the liquid is no greater than
 6. 13. A method of bulkshipping milk comprising the steps of: (a) filling a pressure vessel ofat least 5000 liter capacity at a first location with liquid dairyproduct mixed with CO₂; (b) transporting the filled pressure vessel to asecond location.
 14. The method of claim 13 wherein the product fillingthe pressure vessel has a temperature no greater than 10° C.
 15. Themethod of claim 13 wherein the pressure vessel is thermally insulated.16. The method of claim 15 wherein the R-value of the pressure vessel isat least 27.5.
 17. The method of claim 13 wherein the liquid dairyproduct is selected from the group of raw milk, whole milk, pasteurizedmilk, nonfat milk, reduced fat content milk, and filtered, concentratedor evaporated forms thereof.
 18. The method of claim 13 wherein thepressure vessel is an intermodal tank.
 19. The method of claim 13wherein the pressure vessel is transported by one of the group of rail,truck, and cargo ship.
 20. The method of claim 13 wherein a CO₂ headpressure of at least 68 kPa is maintained in the pressure vessel. 21.The method of claim 13 wherein the liquid dairy product is raw milk. 22.The method of claim 21 wherein the Standard Plate Count of the milk doesnot increase after four days in the pressure vessel.
 23. The method ofclaim 21 wherein the CO₂ is mixed with the milk to achieve a CO₂concentration between about 1750 and 2250 parts per million.
 24. Themethod of claim 21 wherein the Standard Plate Count of the milk does notincrease by more than 1 log₁₀ cfu/ml after four days in the pressurevessel.
 25. The method of claim 23 wherein the Standard Plate Count ofthe milk does not increase by more than 1 log₁₀ cfu/ml after seven daysin the pressure vessel.
 26. The method of claim 21 wherein the counts ofthermoduric bacteria, psychotrophic bacteria and E. coli and coliform donot increase by more than 1 log₁₀ after four days in the pressurevessel.